Process for preparing pharmacologically acceptable salt of n-(1(s)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid

ABSTRACT

There is provided a process for preparing a pharmacologically acceptable salt of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid which comprises condensing an amino acid and N-(1(S) -ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxy-anhydride under basic condition, carrying out decarboxylation under between neutral and acidic condition to obtain N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid, and forming a pharmacologically acceptable salt thereof, wherein the production of a by-product (3):is suppressed by carrying out in an aqueous liquid a series of operations till formation of the pharmacologically acceptable salt or till isolation of the pharmacologically acceptable salt. The present invention enables to prepare the pharmacologically acceptable salt of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid having high quality, in a commercial scale with high yield and economical efficiency.

This application is a divisional of Ser. No. 09/269,107 filed Mar. 19,1999, now U.S. Pat. No. 6,335,453, which was a 371 national phase ofPCT/JP98/03240 filed Jul. 21, 1998.

TECHNICAL FIELD

The present invention relates to a process for economically preparingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid representedby a formula (2):

wherein a group:

is, for example, a group represented by a formula:

or a pharmacologically acceptable salt thereof having high quality, inhigh yield advantageously in a commercial scale. TheN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) and thepharmacologically acceptable salt thereof are very useful compounds asvarious antihypertensive agents, such asN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril)and its maleate (enalapril maleate).

BACKGROUND ART

As a method for obtaining theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) or thepharmacologically acceptable salt thereof, there has been known a methodwherein an N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid(2) is produced starting from an amino acid represented by a formula(1):

wherein a group:

is the same as defined above, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxyanhydriderepresented by a formula (8):

and then a pharmacologically acceptable salt thereof is formedtherefrom. For example, there has been known a method for preparingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline or its maleateas described in Japanese Unexamined Patent Publication No. 48696/1987.

The above-mentioned Japanese Unexamined Patent Publication No.48696/1987 describes a method shown below.

1. N-(1(S)-Ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydrideand 1 to 1.5 time molar amount of L-proline are condensed in a mixedsolvent system of water and an organic solvent having high or lowmiscibility with water under basic condition (preferably pH 9 to 10)and, then, the condensation product is decarboxylated to obtain areaction mixture containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline. Theabove-mentioned patent publication describes that high reaction yield isobtained in a mixed solvent system of water and an organic solventhaving a high miscibility with water such as acetone.

2. The organic solvent having a high miscibility with water such asacetone is distilled away from the above-mentioned reaction mixture, andreplaced with ethyl acetate which is an organic solvent having a lowmiscibility with water, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline is extractedtherewith. In this procedure, the aqueous layer is saturated with sodiumchloride to enhance the extraction efficiency ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline having highwater-solubility.

3. The extraction solution is dehydrated using anhydrous sodium sulfateand, then, the solvent is removed by concentration to obtainN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline.

4. N-(1(S)-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline andacetonitrile are mixed and to this mixture is added maleic acid withheating at 70° C. and the mixture is gradually cooled to obtainN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate.

5. N-(1(S)-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate ispurified by recrystallization from acetonitrile.

However, it was found that methods in whichN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) isproduced from an amino acid (1) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxyanhydride, andthen converted to a pharmacologically acceptable salt thereof, includingthe method described in the above-mentioned publication, potentiallyinclude problems of production of by-products, which causesdisadvantages in yield and quality, a diketopiperazine derivativerepresented by a formula (3):

wherein a group:

is the same as defined above, anN-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-amino acid (hereinafter, alsoreferred to as “carboxy derivative (4)”) represented by a formula (4):

wherein a group:

is the same as defined above, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine represented by aformula (5):

In particular, in the production in a commercial scale requiring longeroperation time, production of a by-product diketopiperazine derivative(3) becomes remarkable, leading to unexpected reduction in the yield ofa desired compound. Furthermore, when by-products, a carboxy derivative(4) and N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5), areproduced, the removal of these compounds is extremely difficult andcauses load in purification.

Moreover, the method described in Japanese Unexamined Patent PublicationNo. 48696/1987 has problems of complicated processes such as use of alarge amount of an extraction solvent, use of various kinds of solvents,replacement of a reaction solvent with an extraction solvent andsaturation of an aqueous layer with an inorganic salt, and problems ofconsumption of longer time, enlargement of the apparatus, increase incost, and the like, due to such complicated processes.

As described above, it is very important to develop a simple method foreconomically preparingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) or apharmacologically acceptable salt thereof having high quality with highyield in a commercial scale.

An object of the present invention is to provide an extremely simplemethod for economically preparing anN-(1((S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) and apharmacologically acceptable salt thereof having high quality with highyield in a commercial scale whereby the production of the by-products,diketopiperazine derivative (3), carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5), is suppressed.

An object of the present invention is, in particular, to provide anextremely simple method for economically preparingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline and itsmaleate having high quality with high yield in a commercial scale, whichmethod solves the above-mentioned problems.

First, it has been found that the production of the by-productdiketopiperazine derivative (3) can be suppressed by carrying out, inthe presence of water, a series of operations for producing anN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) from anamino acid (1) and N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride and forming a pharmacologically acceptable saltthereof and by reason of the protection effect from cyclization reactionby solvation as a protic solvent in addition to the dehydrationsuppressing effect which water essentially possesses, and also byselecting the condition that the N-carbamic acid produced in thereaction system is maintained as a basic salt.

Further it has been found that anN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) having alow content of a diketopiperazine derivative (3), a carboxy derivative(4) and N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) can beobtained by carrying out, in the presence of water under specificreaction conditions, the production of theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) from anamino acid (1) and N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride.

Further it has been found that anN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) and apharmacologically acceptable salt thereof having high quality can beprepared with high yield according to an extremely simple process bycombining the above-mentioned two methods.

Furthermore, it has been found thatN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline can beseparated extremely simply and efficiently, without saturation of anaqueous phase with an inorganic salt and without use of a large amountof extraction solvent, by carrying out extraction and separationoperations under specific temperature condition, especially for theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline having highwater-solubility which requires complicated extraction and separationoperations.

DISCLOSURE OF THE INVENTION

The present invention relates to

(1) a process for preparing a pharmacologically acceptable salt ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid representedby a formula (2):

 wherein a group:

 is a group selected from the group consisting of

 which comprises condensing an amino acid represented by a formula (1):

 wherein a group:

 is the same as defined above, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxyanhydriderepresented by a formula (8):

 under basic condition, decarboxylating a produced carbamic acidderivative under between neutral and acidic condition to obtain anN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2), andforming a pharmacologically acceptable salt thereof, wherein theproduction of a by-product diketopiperazine derivative represented by aformula (3):

 wherein a group:

 is the same as defined above, is suppressed by carrying out in anaqueous liquid a series of operations from the reaction to formation ofthe pharmacologically acceptable salt or a series of operations from thereaction to isolation of the pharmacologically acceptable salt,

(2) the process of the above (1) wherein, in the reaction of producingthe N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2), thecondensation is carried out by gradually adding at least one ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride anda basic pH adjusting agent to an aqueous liquid containing the aminoacid (1) and, if necessary,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydridewith the pH of the aqueous liquid maintained within a range of from 9 to12, and then decarboxylation is carried out to obtain theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) having alow content of a diketopiperazine derivative represented by a formula(3):

 wherein a group:

 is the same as defined above, anN-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-amino acid represented by aformula (4):

 wherein a group:

 is the same as defined above, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine represented by aformula (5):

(3) the process of the above (2) wherein at least one of theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxyanhydride andbasic pH adjusting agent is gradually added over at least ¼ hour,

(4) the process of the above (1), (2) or (3) wherein at least 2 molarequivalents of the amino acid (1) is used based onN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxyanhydride,

(5) the process of the above (1) wherein, in the reaction of producingthe N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2), thereaction is started by addingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxyanhydride toan aqueous liquid containing at least 2 molar equivalents of the aminoacid (1) constituting a basic salt based onN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride,and after the pH of the aqueous liquid reaches a range of from 9 to 12 abasic pH adjusting agent is gradually added to the aqueous liquid tocarry out the condensation with the pH maintained within a range of from9 to 12, and then decarboxylation is carried out to obtain theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) having alow content of a diketopiperazine derivative represented by a formula(3):

 wherein a group:

 is the same as defined above, anN-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-amino acid represented by aformula (4):

 wherein a group:

 is the same as defined above, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-anine represented by a formula(5):

(6) the process of the above (2) or (5) wherein the pH of the aqueousliquid having a pH of from 9 to 12 is maintained within a range of pH10.5±1.0,

(7) the process of the above (1), (2), (3), (4), (5) or (6) wherein theaqueous liquid comprises an organic solvent and water in a weight ratioof from 96:4 to 0:100,

(8) the process of the above (7) wherein the organic solvent is anorganic solvent having a low miscibility with water,

(9) the process of the above (7) or (8) wherein the organic solvent isat least one member selected from the group consisting of a halogenatedhydrocarbon, a fatty acid ester, a ketone and an ether,

(10) the process of the above (2), (3), (4), (5), (6), (7), (8) or (9)wherein, in the condensation reaction of the amino acid (1) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine-N-carboxyanhydride,stirring and mixing are carried out at an agitating power of at least0.1 kW/m³,

(11) the process of any one of the above (1) to (10) wherein thepharmacologically acceptable salt of theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) is formedin a medium comprising an organic solvent and water in a weight ratio offrom 96:4 to 0:100 which medium contains theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2), saidN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) beingseparated from a reaction mixture after the reaction by transferring theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) intoeither one phase in a two-phase medium comprising water and an organicsolvent having a low miscibility with water,

(12) the process of the above (1), (2), (3), (4), (5), (6) or (7)wherein, in the operations of producing theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) andforming the pharmacologically acceptable salt thereof, the series ofoperations from the reaction to formation of the pharmacologicallyacceptable salt or the series of operations from the reaction toisolation of the pharmacologically acceptable salt is carried out in anaqueous liquid essentially consisting of water,

(13) the process of any one of the above (1) to (12) wherein the aminoacid (1) is L-proline and the producedN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) isN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline,

(14) the process of the above (13) wherein using a two-phase liquidcomprising water and an organic solvent having a low miscibility withwater, N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline istransferred into the organic solvent phase by separating the two-phaseliquid at temperature of at least 20° C. or is transferred into thewater phase by separating the two-phase liquid at a temperature of lessthan 20° C., and the pharmacologically acceptable salt thereof is formedin the organic solvent phase or the water phase and, if necessary,isolated,

(15) the process of the above (13) or (14) wherein the pharmacologicallyacceptable salt thereof is maleic acid salt thereof,

(16) the process of the above (15) wherein steps for forming andcrystallizing the salt are carried out in an aqueous liquid essentiallyconsisting of water in which an inorganic salt coexists,

(17) the process of the above (13), (14), (15) or (16) wherein themaleic acid salt thereof is formed by gradually adding an aqueous liquidcontaining N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline toan aqueous liquid containing maleic acid,

(18) the process of the above (15), (16) or (17) wherein the steps forforming and crystallizing the salt are carried out at from 40 to 70° C.

(19) the process of any one of the above (1) to (18) wherein at least anequimolar amount of water exists based on a producedN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2), in theseries of operations from the reaction of producing theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) toformation of the pharmacologically acceptable salt or the series ofoperations from the reaction to isolation of the pharmacologicallyacceptable salt,

(20) a process for preparingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleatewherein a process in which steps for forming and crystallizing a salt inan aqueous liquid containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline and maleicacid are carried out in an aqueous liquid essentially consisting ofwater, is carried out in the coexistence of an inorganic salt and/or atfrom 40 to 70° C.,

(21) the process of the above (20) wherein the process is carried outusing a reaction mixture after production ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline,

(22) a process for preparing a pharmacologically acceptable salt ofN-(1((S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid representedby a formula (2):

 wherein a group:

 is a group selected from the group consisting of

 which comprises forming a pharmacologically acceptable salt ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid from anN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) containedin a reaction mixture after production of theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) and, ifnecessary, isolating the pharmacologically acceptable salt, wherein theproduction of a by-product diketopiperazine derivative represented by aformula (3):

 wherein a group:

 is the same as defined above, is suppressed by carrying out anoperation in a medium comprising an organic solvent and water in whichthe proportion of water is higher than a weight ratio of the organicsolvent/water of 96/4,

(23) the process of the above (22) wherein the pharmacologicallyacceptable salt is formed in the organic solvent phase in which watercoexists and which is obtained by extracting or washing the reactionmixture containing theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) and, ifnecessary, the pharmacologically acceptable salt thereof is isolated,

(24) the process of the above (22) or (23) wherein at least an equimolaramount of water exists based on theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) in theoperations of forming and, if necessary, isolating the pharmacologicallyacceptable salt of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-aminoacid (2),

(25) the process of the above (22), (23) or (24) wherein theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) isN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline,

(26) the process of the above (22), (23), (24) or (25) wherein thepharmacologically acceptable salt is a maleic acid salt,

(27) the process of the above (22), (23), (24), (25) or (26) whereinsteps for forming and crystallizing the salt are carried out at from 40to 70° C.,

(28) a process for separatingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline wherein, byseparating a two-phase medium comprising water and an organic solventhaving a low miscibility with water which medium containsN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline, at atemperature of at least 20° C.,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline is transferredinto the organic solvent phase or, by separating the two-phase medium ata temperature of less than 20° C.,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline is transferredinto the water phase,

(29) the process of the above (28) wherein the organic solvent is anacetic acid ester,

(30) the process of the above (28) or (29) wherein, in the process fortransferring N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolineinto the organic solvent phase, the transfer is carried out withoutsaturating the water phase with an inorganic salt,

(31) a process for preparingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline whichcomprises producingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline from L-prolineand N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride, wherein condensation is carried out by graduallyadding at least one of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride and a basic pH adjusting agent to an aqueous liquidcontaining L-proline and, if necessary,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride,with the pH of the aqueous liquid maintained within a range of from 9 to12, and then decarboxylation is carried out under between neutral andacidic condition to obtainN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline having a lowcontent of a diketopiperazine derivative represented by a formula (6):

 wherein all asymmetric carbon atoms with * have (S)-configuration,N-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-L-proline represented by aformula (7):

 wherein all asymmertric carbon atoms with * have (S)-configuration, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine represented by aformula (5):

(32) the process of the above (31) wherein at least one of theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride andthe basic pH adjusting agent is added over at least ¼ hour,

(33) the process of the above (31) or (32) wherein at least 2 molarequivalents of L-proline is used based onN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride,

(34) a process for preparingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline whichcomprises producingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline from L-prolineand N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride, wherein a reaction is started by addingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxyanhydride toan aqueous liquid containing at least 2 molar equivalents of L-prolineconstituting a basic salt based onN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride,and after the pH of the aqueous liquid reaches a range of from 9 to 12 abasic pH adjusting agent is gradually added to the aqueous liquid tocarry out a condensation with the pH maintained within a range of from 9to 12, and then decarboxylation is carried out under between neutral andacidic condition to obtainN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline having a lowcontent of a diketopiperazine derivative represented by a formula (6):

 wherein all asymmetric carbon atoms with * have (S)-configuration,N-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-L-proline represented by aformula (7):

 wherein all asymmertric carbon atoms with * have (S)-configuration, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine represented by aformula (5):

(35) the process of the above (31), (32), (33) or (34) wherein theaqueous liquid comprises an organic solvent and water in a weight ratioof from 96:4 to 0:100,

(36) the process of the above (31), (32), (33), (34) or (35) wherein, inthe reaction of L-proline andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxyanhydride,stirring and mixing are carried out at an agitating power of at least0.1 kW/m³,

(37) the process of the above (35) or (36) wherein the organic solventis at least one member selected from the group consisting of ahalogenated hydrocarbon, a fatty acid ester, a ketone and an ether,

(38) the process of the above (31), (32), (33), (34), (35) or (36)wherein the reaction of producingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline is carried outin an aqueous liquid essentially consisting of water,

(39) a process for purifying a pharmacologically acceptable salt ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid representedby a formula (2):

, wherein a group:

is a group selected from the group consisting of

 wherein the purification is carried out in an aqueous liquid wherebythe production of a by-product diketopiperazine derivative representedby a formula (3):

 wherein a group:

 is the same as defined above, is suppressed and also theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) andpharmacologically acceptable salt thereof having a low content of anN-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-amino acid represented by aformula (4):

 wherein a group:

 is the same as defined above, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine represented by aformula (5):

 is obtained,

(40) the process of the above (39) wherein the aqueous liquid is amedium comprising an organic solvent and water in which the proportionof water is higher than a weight ratio of the organic solvent/water of96/4,

(41) the process of the above (39) or (40) wherein the purification ofthe pharmacologically acceptable salt is carried out at from 40 to 70°C.

(42) a process for purifyingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate whichcomprises carrying out the purification ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate in anaqueous liquid essentially consisting of water, wherein the maleate ispurified in the coexistence of an inorganic salt and/or at from 40 to70° C. whereby the production of a by-product diketopiperazinederivative represented by a formula (6):

 wherein all asymmetric carbon atoms with * have (S)-configuration, issuppressed and alsoN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate havinga low content of N-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-L-prolinerepresented by a formula (7):

 wherein all asymmertric carbon atoms with * have (S)-configuration, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine represented by aformula (5):

 is obtained, and

(43) the process of the above (39), (40), (41) or (42) wherein thepurification method is recrystallization or reslurry washing.

BEST MODE FOR CARRYING OUT THE INVENTION

The amino acid in the present invention represented by the formula (1):

is an amino acid wherein the group:

in the formula is a group selected from the group of an imino acidresidue, preferably of a cyclic imino acid residue. In the case of animino acid residue, R¹ is an alicyclic monocyclic or bicyclic serieshaving 5 to 10 ring members and R² is hydrogen atom. Representativeexamples of the imino acid residue are, for example, a group representedby a formula:

and the like.

In the case of a cyclic imino acid residue, R¹ and R² are combined and,together with nitrogen and carbon atoms to which R¹ and R² areconnected, form a heterocyclic monocyclic or bicyclic series having 5 to10 ring members. The above-mentioned cyclic imino acid residue is, forexample, a residue of proline or proline analogue, or a group derivedfrom them and, in the above-mentioned group, the pyrrolidine ring can beexchanged for, for example, piperidine ring, quinuclidine ring,isoindoline ring, N-alkylimidazolidine ring, octahydroindole ring,octahydroisoindole ring, decahydroquinoline ring, decahydroisoquinolinering, 1,2,3,4-tetrahydroisoquinoline ring and similar ring thereto.These rings may be substituted or linked by an oxo group, hydroxylgroup, mercapto group, an alkylmercapto group, an alkoxy group, an alkylgroup or the like. Representative examples of the cyclic imino acidresidue are groups represented by formulae:

and the like.

Among these amino acids, for example, L-proline and 1, 2,3,4-tetrahydro-3-isoquinolinecarboxylic acid are commercially available.1,2,3,4-Tetrahydro-6,7-dimethoxy-3-isoquinolinecarboxylic acid isobtained by a method described in, for example, U.S. Pat. No. 4,912,221and 1,4-dithia-7-azaspiro[4,4]nonane-8-carboxylic acid is obtained by amethod described in, for example, U.S. Pat. No. 4,468,396. Further,1-methyl-2-oxo-4-imidazolidine-carboxylic acid,octahydrocyclopenta[b]pyrrole-2-carboxylic acid and2-azabicyclo[2,2,2]octane-3-carboxylic acid are obtained, for example,by methods described in Int. J. Pept. Protein Res., 33(6), 403-11(1989),Tetrahedron Lett., 34(41), 6603-6(1993) and Tetrahedron Lett., 33(48),7369-72(1992), respectively.

The N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydrideused in the present invention can be prepared according to methodsdescribed in, for example, Japanese Unexamined Patent Publication No.48696/1987, U.S. Pat. Nos. 4,686,295 and 5,359,086, and the like. Forexample, the N-carboxyanhydride can be easily prepared approximatelyquantitatively by adding a phosgene solution or introducing a phosgenegas to N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) or itsinorganic acid salt such as a hydrochloride in an organic solvent and byreacting them with heating. TheN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydrideproduced by the reaction can be usually used in the form of a reactionsolution as obtained without particular purification after removal ofthe remaining phosgene and hydrogen chloride, or can also becrystallized for use. In the case of a reaction solution, it isadvantageous to use an organic solvent having a low miscibility withwater as a reaction solvent so that in the present invention it can besuitably used as it is. In the case of being crystallized for use, thesubsequent present invention can be carried out without using an organicsolvent at all.

In the N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid in thepresent invention represented by the formula (2):

a group:

is the same as defined above. The above-mentioned imino acid residue andcyclic imino acid residue contribute to exhibiting of excellentantihypertensive action. When in the formula (2) the carbon atom towhich a carboxyl group is connected is an asymmetric carbon atom, thecompound (2) in which this carbon atom has (S)-configuration isgenerally useful as an antihypertensive agent. When another asymmetriccarbon atom exists, one having desired configuration based on thisasymmetric carbon atom can be used. In the case that the above-mentionedgroup:

is a group represented by a formula:

particularly desirable configuration thereof is, for example,

When the above-mentioned cyclic imino acid residue is a proline residue,particularly a L-form (namely, (S)-configuration) proline residue, theproduct (2) is enalapril, a particularly useful antihypertensive agent.

Examples of the pharmacologically acceptable salt ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) obtainedby the present invention include an inorganic acid salt such ashydrochloride, sulfate or phosphate, an organic acid salt such asacetate, maleate, fumarate, tartarate or citrate, an amino acid adductsuch as glycine or phenylalanine.

The aqueous liquid means a solution in which water coexists, andexamples of the aqueous liquid include water or a mixture of an organicsolvent and water. The above-mentioned organic solvent may be either anorganic solvent having a high miscibility with water or an organicsolvent having a low miscibility with water.

Examples of the organic solvent having a high miscibility with waterinclude, for example, acetone, acetonitrile, tetrahydrofuran (THF),dioxane, a mixture thereof and the like. Among these, acetone andtetrahydrofuran are preferred from viewpoints of easy handling andsafety of the solvent. This organic solvent having a high miscibilitywith water means a solvent that when mildly mixed with the same volumeof pure water generally at 20° C. under one atmosphere, uniformappearance of the mixture remains even after the flow ceases.

Examples of the organic solvent having a low miscibility with waterinclude, for example, a halogenated hydrocarbon such as methylenechloride, chloroform or 1,2-dichloroethane; a fatty acid ester such asmethyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate,n-butyl acetate, isobutyl acetate, methyl propionate or ethylpropionate; a ketone such as methyl ethyl ketone, methyl n-propylketone, diethyl ketone or methyl isobutyl ketone; a hydrocarbon such astoluene or n-hexane; an ether such as diethyl ether, dipropyl ether,diisopropyl ether, dibutyl ether or methyl tert-butyl ether; a mixturethereof; and the like. Among these, a halogenated hydrocarbon, a fattyacid ester, a ketone and an ether are preferred from viewpoint ofsolubility for N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride, and the like, and among them, a fatty acid ester,particularly an acetate, more particularly ethyl acetate, is preferredfrom viewpoints of easy handling, safety of the solvent, cost of thesolvent, use advantage as a solvent in extraction and salt formation,high effect for stabilizingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) (effectfor suppressing the production of a by-product diketopiperazinederivative (3)) and the like. This organic solvent having a lowmiscibility with water means an organic solvent other than theabove-mentioned organic solvent having a high miscibility with water.

Further, the above-mentioned organic solvents can be used together. Forexample, the organic solvent having a high miscibility with water andthe organic solvent having a low miscibility with water can be usedtogether.

The ratio of the organic solvent to water in the above-mentioned aqueousliquid differs depending on solubilities of a reactant (1) and a product(2). The weight ratio is from 96:4 to 0:100, generally from 20:1 to0:100 and is normally from 10:1 to 0:100 from viewpoint of productivity.When the amino acid (1) is L-proline and the product (2) isN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril),the weight ratio of the organic solvent to water may also be from 96:4to 0:100, and is preferably from 20:1 to 0:100, particularly from 10:1to 0:100 from viewpoint of productivity. The aqueous liquid may be anaqueous liquid essentially consisting of water.

The “aqueous liquid essentially consisting of water” in the presentinvention means an aqueous system which may contain an organic solventto an extent that in the reaction and salt formation, a resulting effectis nearly the same with an effect given when carrying out the reactionand salt formation in a water alone. A ratio of water contained thereinvaries depending on the kind of an organic solvent used and steps.

It is desirable that the amount of water is normally at least equivalentmole, preferably at least 2-fold mole, more preferably at least 3-foldmole, further preferably at least 4-fold mole, based on the product (2),since the production of a by-product diketopiperazine derivative (3) issuppressed. For example, the production of the by-product (3) can besuppressed to at most ½ to ⅓, preferably to almost negligible level asdescribed in Examples 8 and 10 shown below.

In the process of the present invention,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride andan amino acid (1) are first condensed in an aqueous liquid under basiccondition to obtain a carbamic acid derivative.

Examples of the basic pH adjusting agent used in the present inventionfor carrying out the condensation reaction under basic conditioninclude, for example, an inorganic base such as a hydroxide, carbonateor hydrogencarbonate of alkaline metal or alkaline earth metal, and anorganic base such as a secondary amine, tertiary amine or quaternaryammonium hydroxide. Concrete examples thereof include, for example, analkaline metal hydroxide such as sodium hydroxide, potassium hydroxideor lithium hydroxide; an alkaline metal carbonate such as sodiumcarbonate, potassium carbonate or lithium carbonate; an alkaline metalhydrogencarbonate such as sodium hydrogencarbonate or potassiumhydrogencarbonate; an alkaline earth metal hydroxide such as magnesiumhydroxide or calcium hydroxide; a secondary amine such as dimethylamine,diethylamine, diisopropylamine or dicyclohexylamine; a tertiary aminesuch as triethylamine, tripropylamine, tributylamine, triamylamine,pyridine or N-methylmorpholine; a quaternary ammonium hydroxide such astetramethyl-, tetraethyl-, tetrapropyl-, tetrabutyl-, tetraamyl-,tetrahexyl- or benzyltrimethyl-ammonium hydroxide. However, the basic pHadjusting agent is not limited to those examples.

As to the basic pH adjusting agent, an inorganic base, particularly analkaline metal hydroxide such as sodium hydroxide, potassium hydroxideor lithium hydroxide is preferred and, further, sodium hydroxide andpotassium hydroxide are preferred, from viewpoints of inexpensiveness,easy handling and easy disposal of waste water. The above-mentionedinorganic base is preferably used in the form of an aqueous solutionthereof from viewpoint of operability, and normally, it is advantageousthat the base is used, for example, in the form of a 2 to 20 N,preferably 5 to 20 N aqueous solution of an alkaline metal hydroxide.The above-mentioned basic pH adjusting agent may be used alone and mayalso be used in admixture of two or more thereof.

The amount used of the above-mentioned basic pH adjusting agent is anamount necessary for maintaining an aqueous liquid at specific basicity.

An aqueous liquid is adjusted to basic with the above-mentioned basic pHadjusting agent, however, there is no need to add a base separately foradjusting the aqueous liquid to basic. There can be used, as the aqueousliquid, an aqueous liquid which has been made to have a pH bufferingaction by adding disodium hydrogenphosphate, hydrochloric acid, boricacid or the like, and a surfactant, a phase-transfer catalyst etc. mayalso be added if necessary.

For securing a high yield and high quality consistently in the presentreaction, it is important to suppress side reactions such as productionof a carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) due to hydrolysisin reacting and to allow the main reaction to proceed smoothly. Forthis, it is beneficial to carry out the condensation at pH 9 to 12 withgradually adding at least one ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride anda basic pH adjusting agent to an aqueous liquid containing an amino acid(1) and, if necessary, N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride. By this, it is possible to suppress the productionof by-products to at most ½ to ⅓ as a whole and reduce the content ofthe by-products (3) to (5) in the product (2) to concretely less than 5%by weight, preferably less than 2% by weight, such as in Example 7described below. Therefore, the yield of the desired product (2) can beraised to a level of 95% or more.

In this case, increase in the amount of the amino acid (1) used is alsoeffective for smoothly proceeding the main reaction with suppressing aside-reaction such as hydrolysis. Specifically, use of at least 2 molarequivalents of an amino acid (1) based onN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride ispreferable since it further enhances the effect.

The time for addition of at least one ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride anda basic pH adjusting agent is, in terms of time for addition of wholeamount, generally at least ¼ hour, normally at least ⅓ hour, preferablyat least ½ hour, and there is no upper limit, however, it is generallyat most 20 hours, normally at most 15 hours, preferably at most 10hours, from viewpoint of productivity and the like. As the gradualaddition method, there can be employed, for example, a method in whichmaterials are added at constant rate, a method in which materials areadded portionwise, and the like, and particularly, the method in whichmaterials are added at constant rate is preferably employed fromviewpoint of improvement in yield and quality, operability and the like.The N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydridecan be added, for example, after mixing or dissolving in a solvent usedin the above-mentioned reaction before addition, or can be added as itis in the form of a powder.

By this, generally the reaction can be suitably carried out not only ina mixed solvent system of water and an organic solvent having a highmiscibility with water but also in a mixed solvent system of water andan organic solvent having a low miscibility with water. And in thereaction in an aqueous liquid essentially consisting of water in whichthe proportion of water in an aqueous liquid used is increased and theeffect for suppressing the production of the diketopiperazine derivative(3) is maximized, there is a tendency that the reaction rate of mainreaction decreases and the influence by side-reaction due to theelongation of reaction time increases. Therefore, it is suitable forsolving the above-mentioned problems that the proportion constituting abasic salt as an active species of the amino acid (1) used is increased,and specifically, N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride is added to an aqueous liquid in which said activespecies exists in an amount of at least 2 molar equivalents based on theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxyanhydride forreaction. This condition also includes a case in which the pH of anaqueous liquid at the start of the reaction is over 12. However, whencondensation reaction is effected with maintaining the pH within a rangeof from 9 to 12 by gradually adding a basic pH adjusting agent to areaction liquid after the pH reaches the range from 9 to 12 as a resultof steep reduction of the pH accompanying smooth proceeding of the mainreaction, the N-carbamic group produced is maintained as its basic saltand the amount of the active species of the above-mentioned amino acid(1) is always maintained at maximum, and thereby the reaction time canbe shortened and the production of the carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) owing to hydrolysiscan be minimized. By this, the reaction can be carried out particularlysuitably even in the case that reaction field is substantially water.

Advantageously the range of the pH maintained at 9 to 12 is preferablywithin the range of pH 10.5±1.0, more preferably within the range of pH10.5±0.5.

When the pH in reaction is out of the above-mentioned range, the totalamount of the by-products tends to increase. When the pH is lower, themain reaction does not easily proceed, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) tends to beproduced due to hydrolysis of the carboxyanhydride moiety. On the otherhand, when the pH is higher, a carboxy derivative (4) tends to beproduced due to hydrolysis of the ethoxycarbonyl moiety.

Regarding the reaction, since there is a tendency that the reactionsystem becomes heterogeneous system of liquid-liquid or solid-liquidparticularly in a mixed solvent system of water and an organic solventhaving a low miscibility with water or in an aqueous liquid essentiallyconsisting of water, it is preferable to suitably stir and mix thereaction system so as to obtain sufficient dispersion. In this case, theagitating power is generally at least 0.1 kW/m³, and advantageously itis preferably at least 0.2 kW/m³, more preferably at least 0.5 kW/m³from viewpoint of improvement in quality and yield. There is notparticular upper limit, however, it is at most 5 kW/m³ from practicalaspect of the stirrer. Therefore, it can be favorably selected withinthe range generally from 0.1 to 5 kW/m³, and normally from 0.5 to 3kW/m³.

In the present invention, the molar ratio of an amino acid (1) toN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride isgenerally from 0.5 to 5, however, from viewpoint of the quality andproductivity of the resultingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2), themolar ratio is generally at least 0.7, normally at least 1. As describedabove, it is 5 suitable that the molar ratio is at least 2 forexhibiting the maximum effect of the present invention. The upper limitthereof is not particularly limited and it is advantageous to begenerally at most 5, normally at most 4, particularly at most 3 fromviewpoints of economy, load on disposal of waste water, and the like.

The amino acid (1) is preferably added in a whole amount thereof fromthe start from viewpoint of securing of high yield, simple operabilityand the like.

The charging concentration of an amino acid (1) is, in terms of thewhole amount of the amino acid (1) based on an aqueous liquid, generallyfrom about 5 to about 200% (w/v), though it changes depending on thekind of the amino acid (1). The higher concentration is moreadvantageous from viewpoints of yield, quality, reaction rate andproductivity, and a mixed solvent system of water and an organic solventhaving a low miscibility with water and an aqueous liquid essentiallyconsisting of water have higher solubility for a water-soluble substanceas compared with a mixed solvent system of water and an organic solventhaving a high miscibility with water. Therefore, it has been found thatthe reaction can be advantageously carried out at a high concentrationof at least 10% (w/v), preferably at least 20% (w/v), more preferably atleast 30% (w/v) in the case of using, as the solvent for the aqueousliquid, a mixed solvent system of water and an organic solvent having alow miscibility with water or an aqueous liquid essentially consistingof water. For example, in the case of preparingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril),it is also possible to carry out the reaction at a high concentration ofat least 100% (w/v) as a total amount of L-proline to the aqueousliquid.

The reaction temperature is a temperature at which the reaction mixtureis not frozen, normally at most 60° C., preferably at most 50° C., morepreferably at most 40° C. When the reaction temperature is too high, aside reaction increases whereby yield and quality tend to lower. Forexample, the production ofN-1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril) isfavorably carried out generally at a temperature within a range of25±15° C.

After the condensation reaction ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride andan amino acid (1), the resulting carbamic acid derivative is decomposed(decarboxylated) under between neutral and acidic condition to producean N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2).

The decarboxylation is carried out in an aqueous liquid and the reactioneasily proceeds by mixing an acid with the reaction mixture. Thedecarboxylation is carried out at a temperature at which the reactionmixture is not frozen, normally at most 60° C., preferably at most 50°C., more preferably at most 40° C. When the temperature indecarboxylation is too high, a side reaction increases whereby yield andquality tend to lower. Accordingly, it is generally advantageous tocarry out the reaction at a low temperature of, for example, at most 20°C., preferably at most 10° C., in order to reduce the production of aby-product diketopiperazine derivative (3).

Preferably, the decarboxylation is carried out under between neutral andacidic condition paying attention to generated heat and foaming, whichdiffers in the reaction concentration. It is advantageous to adjust thepH of the reaction mixture to normally at most pH 8, preferably at mostpH 7, from viewpoint of a decarboxylation rate. And, there is no need tobe strongly acidic and generally the pH can be freely selected within arange of from 1 to 6. The pH is finally adjusted to pH 4 to 5, which isa pH near an isoelectric point ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2), toproduce an N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid(2).

The acid used in the decarboxylation reaction is not particularlylimited, and preferably it is a strong acid from viewpoint of practicaluse. Normally a mineral acid such as hydrochloric acid or sulfuric acidis preferable. Hydrochloric acid is more preferable and conc.hydrochloric acid is most preferable. These may be used alone or inadmixture thereof. The amount of the acid is such an amount as requiredto neutralize a basic component in order to adjust the pH to anisoelectric point of anN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2). The acidcan be added to an aqueous liquid containing a reaction mixture, payingattention to generated heat and foaming, or to the acid can be added anaqueous liquid containing a reaction mixture.

The use of preferred basic component and acidic component in between thecondensation and the decarboxylation reactions contributes to productionof an inorganic salt which is easy of disposal and to improvement ofextraction efficiency ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) due to asalting-out effect of the produced inorganic salt. An inorganic saltsuch as sodium chloride or pottassium chloride has excellent salting-outeffect.

In the resulting reaction mixture, the production of a by-productdiketopiperazine derivative (3) is suppressed by the presence of waterand the N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2)is stabilized. Preferably, the reaction mixture is quickly subjected tothe subsequent step.

According to the reaction process of the present invention, theproduction of by-products is suppressed in the reaction not only in amixed solvent system of water and an organic solvent having a highmiscibility with water, which has hitherto been regarded as a favorablesolvent, but also in a mixed solvent system of water and an organicsolvent having a low miscibility with water or in an aqueous liquidessentially consisting of water. Therefore, higher increase in yield ofthe product (2) can be expected. For example, in the production ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril),the reaction yield of at least about 95% can be expected in any of theabove-mentioned solvent systems. Such an establishment of higher yield(reduction in impurities) highly contributes to obtaining high qualityof an N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) orpharmacologically acceptable salt thereof. Further, as described below,if a mixed solvent system of water and an organic solvent having a lowmiscibility with water or an aqueous liquid essentially consisting ofwater is suitably used, simplification or omission of extractionoperation becomes possible such as no need to replace a solvent in thesubsequent extraction operation.

The case for producingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril)will be described below.

When N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline(enalapril) is produced from L-proline andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline having a lowcontent of a diketopiperazine derivative represented by the formula (6):

wherein all asymmetric carbon atoms with * have (S)-configuration,N-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-L-proline represented by theformula (7):

wherein all asymmetric carbon atoms with * have (S)-configuration, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine represented by theformula (5):

can be obtained by carrying out a condensation by gradually adding atleast one of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride and a basic pH adjusting agent to an aqueous liquidcontaining L-proline and, if necessary,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydridewith the pH of the aqueous liquid maintained within a range from 9 to 12and carrying out a decarboxylation under between neutral and acidiccondition.

At least one of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine-N-carboxyanhydride and a basic pH adjusting agent is desirably addedover at least ¼ hour. Though there is not particular upper limit, it isat most 20 hours from viewpoint of productivity and the like.

In the reaction in an aqueous liquid essentially consisting of water inwhich the effect for suppressing the production of a by-productdiketopiperazine derivative (3) is maximized,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline having a lowcontent of a diketopiperazine derivative (6),N-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-L-proline (7) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) can be favorablyobtained by a process thatN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxyanhydride isadded to an aqueous liquid which contains at least two molar equivalentsof L-proline constituting a basic salt based onN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride, tostart the reaction, and condensation is carried out, with maintainingthe pH value within a range of from 9 to 12, by gradually adding a basicpH adjusting agent after the pH of the aqueous liquid reaches a range offrom 9 to 12 and, then, decarboxylation is carried out.

The above-mentioned aqueous liquid is a medium comprising an organicsolvent and water in a weight ratio of 96:4 to 0:100, preferably 20:1 to0:100, more preferably 10:1 to 0:100. The organic solvent isappropriately selected from the above-mentioned organic solvents havinga high miscibility with water and organic solvents having a lowmiscibility with water, and particularly a halogenated hydrocarbon, afatty acid ester, a ketone and an ether are preferable. Theabove-mentioned aqueous liquid may also be substantially water.

The maintained pH of the aqueous liquid in the condensation reaction iswithin a range of from 9 to 12, preferably within a range of 10.5±1.0,more preferably within a range of 10.5±0.5. By maintaining the pH withinthe above-mentioned range, the content of the by-products (5) to (7) inthe resulting enalapril can be reduced, specifically to less than 5% byweight, preferably to less than 2% by weight.

In the condensation reaction, stirring and mixing are preferably carriedout at an agitating power of at least 0.1 kW/m³. Though there is notparticular upper limit, it is at most 5 kW/m³ from viewpoint ofpractical aspect of a stirrer.

Other conditions such as the amount of water to enalapril, the amount ofL-proline to N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride, the charging concentration of L-proline, the kindand amount of a basic pH adjusting agent, and an acid, reactionconditions and reaction method used for a decarboxylation reaction arethe same as described for the above-mentioned production of the compound(2).

Thus obtained reaction mixture containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) after theabove-mentioned decarboxylation reaction can be used as it is forformation of a pharmacologically acceptable salt, and alternatively theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) can beonce separated before formation of a pharmacologically acceptable salt,taking into consideration of the removal of water-soluble impurities.

Then, a process for separating theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) from areaction mixture after the decarboxylation reaction will be describedbelow.

For the separation ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) existingin the reaction mixture after the decarboxylation reaction,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) isextracted into an organic solvent using a two-phase medium of theorganic solvent and water near isoelectric pH ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2). Theisoelectric points ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) somewhatdiffer individually, and is usually near pH 4 to 5. At the isoelectricpH, the solubility ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) into anaqueous solution is minimum. For enhancing an extraction efficiency, itis beneficial to carry out the extraction at a pH within the range ofisoelectric point ±2, preferably isoelectric point ±1, more preferablyisoelectric point ±0.5. The extraction is carried out in a two-phasesystem of a water phase and an organic solvent phase, and it isbeneficial to remove water-soluble impurities such as the remainingamino acid (1) and produced inorganic salt by transferring them into awater phase and to transferN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) into anorganic solvent phase.

The organic solvent used for the extraction is a solvent capable offorming the organic solvent phase constituting a two-phase systemtogether with the water phase, and usually an organic solvent having alow miscibility with water which can be used in the production reactionof the compound (2) is preferably used. As the organic solvent having alow miscibility with water, a halogenated hydrocarbon, a fatty acidester, a ketone and an ether are preferred as described above, and amongthese, a fatty acid ester, particularly an acetate, more particularlyethyl acetate is preferable from viewpoints of easy handling, safety ofthe solvent, cost of the solvent, use advantage as a solvent in theformation of a pharmacologically acceptable salt, high effect forstabilizing N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid(2) (effect for suppressing the production of diketopiperazinederivative (3)) and the like.

Therefore, when the production reaction of the compound (2) in thepresent invention is carried out in a two-phase medium of water and anorganic solvent having a low miscibility with water, it is possible thatthe water phase is separated with carrying out the decarboxylation orafter the decarboxylation to obtain a solution comprising the organicsolvent used for the production reaction ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2). By thisthere can be simply and efficiently obtained an organic solvent phasecontaining N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid(2). The N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2)remaining in the water phase can be recovered, if necessary, byextracting with the above-mentioned organic solvent. Further, theresulting organic solvent phase can be washed with water, if necessary.

When the amount of water in an aqueous liquid containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) iscontrolled through this extraction and separation operation so that theweight ratio of organic solvent to water is 96:4 as a whole or theproportion of water is higher than the above ratio, the production ofby-products such as a diketopiperazine derivative (3) can be suppressed.By this, the production of by-products can be at least suppressed to atmost ½ to ⅓ such as in Examples 8 and 10 described below. Needless tosay, this suppression effect is higher when the water content is higher,and the proportion of water is higher preferably than the weight ratioof organic solvent to water of 96:4.

The above-mentioned extraction operation and washing operation arecarried out at a temperature of at most the boiling point of a solventand at which the solvent is not frozen though the temperature depends onthe kind of the solvent and operation time. Particularly highertemperature is not required, and the operations are carried out,practically at a temperature at which the solution is not frozengenerally at most 60° C., normally at most 50° C., preferably at most40° C. For minimizing production of a by-product diketopiperazinederivative (3), it is generally advantageous to carry out theabove-mentioned operations, for example, at a lower temperature of atmost 20° C., preferably at most 10° C.

However, it has been found that especially when theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) isN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril)having high water-solubility, partition into an organic solvent phasecan be accomplished extremely efficiently by carrying out theabove-mentioned operations at a higher temperature. For example, thepartition ratio ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril) ina two-phase system of a water phase and an organic solvent phase highlydepends on temperature, and higher partition ratio into an organicsolvent phase is obtained at a temperature range of at least 20° C.,preferably at least 25° C., more preferably at least 30° C. Theimprovement of this partition ratio into an organic solvent phase at ahigher temperature exerts a large effect especially when the amount ofan organic solvent is small and when using an organic solvent revealingnot necessarily excellent extraction efficiency forN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril),for example, an acetate such as ethyl acetate.

The upper limit of the above-mentioned operation temperature is variablewith an operation mode such as continuous extraction or batchwiseextraction and is not particularly restricted, however, it is notparticularly required to be higher, and is practically at most 60° C.,normally at most 50° C., preferably at most 40° C.

Conventionally, in extractingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril),extremely troublesome operations are required such as utilization ofsalting-out effect and use of a large amount of an extraction solventdue to high water-solubility of enalapril. However, according to thepresent invention, a solution of an organic solvent containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril)can be simply and efficiently obtained in high yield and high quality bycarrying out the operation under the above-mentioned temperaturecondition, without a treatment for saturating a water phase with a largeamount of a salt and without a multiple extraction with a large amountof an organic solvent.

When the reaction is carried out using an organic solvent having a highmiscibility with water, the organic solvent is removed and thensubstituted with an organic solvent having a low miscibility with waterand the same operations such as washing with water are carried out underthe same condition.

Further, by carrying out an operation at a low temperature, normally ata temperature of less than 20° C., preferably at a temperature of lessthan 10° C. in a two-phase medium of water and an organic solvent havinga low miscibility with water by utilizing the above-mentioned partitionbehavior, it is possible to efficiently partitionN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril)reversely into the water phase, and to efficiently extract impuritieshaving a low water-solubility such as a diketopiperazine derivative (3)into the organic phase by washing the water phase with an organicsolvent. In this case, there is preferably used a method in whichN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril) isextracted into water from the above-mentioned solution of an organicsolvent containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril)from which the water-soluble impurities have been removed.

Namely, if a two-phase medium of water and an organic solvent having alow miscibility with water containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline is separatedat a temperature of at least 20° C.,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline can betransferred into the organic solvent phase, or if the two-phase mediumis separated at a temperature of less than 20° C.,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline can betransferred into the water phase. Thus,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline can beseparated. Especially, the case in which the organic solvent is anacetate is preferable from the viewpoints of easy handling, safety ofthe solvent, and high effect for stabilizingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline. Further, evenwhen the above-mentioned two-phase medium is separated at a temperatureof at least 20° C.,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline can betransferred into the organic solvent phase without saturating the waterphase with an inorganic salt.

Then the explanation is given below as to an operation, which is carriedout in the coexistence of water, for forming a pharmacologicallyacceptable salt fromN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) in areaction mixture, extracted solution or washing solution containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2).

The existence of water contributes to not only suppression of theproduction of a by-product diketopiperazine derivative (3) fromN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2), but alsoto suppression of the production of a by-product diketopiperazinederivative (3) from the resulting pharmacologically acceptable salt.

A reaction mixture, extracted solution or washing solution containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) obtainedby another procedure can also be used for the formation of apharmacologically acceptable salt.

During the formation and separation operation of this salt, the weightratio of an organic solvent and water is controlled so as to be in therange from 96:4 to 0:100 as in the above-mentioned reaction andseparation operation. Dehydration of a solution containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) byaddition of a dehydrating agent or concentration operation ratherinvites disadvantage of production of a by-product diketopiperazinederivative (3).

In the present invention, the following procedures are alsocontemplated:

a pharmacologically acceptable salt ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) is formedin a medium comprising an organic solvent and water in a weight ratio of96:4 to 0:100 which medium containsN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino. acid (2), whichN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) has beentransferred into either one phase and separated by extracting from areaction mixture after reaction containing the product (2) in the manneras described above using a two-phase medium comprising water and anorganic solvent having a low miscibility with water,

a pharmacologically acceptable salt ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) is formedin an organic solvent phase in which water coexists and which phase isobtained by extracting or washing a reaction mixture containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) using amedium comprising an organic solvent and water in which a weight ratioof the organic solvent: water is from 96:4 to 0:100, or in a water phaseobtained by extracting or washing a reaction mixture containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) using theabove-mentioned medium, and, if necessary, the pharmacologicallyacceptable salt is isolated, and using a two-phase liquid comprisingwater and an organic solvent having a low miscibility with water,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline is transferredinto the organic solvent phase by separating the two-phase liquid at atemperature of at least 20° C., or is transferred into the water phaseby separating the two-phase liquid at a temperature of less than 20° C.and a pharmacologically acceptable salt thereof is formed in the organicsolvent phase or the water phase, and isolated.

The solvent used for the formation of the pharmacologically acceptablesalt is basically the same as that used for the production reaction ofthe compound (2). The preferable kind of the solvent and the ratio ofthe organic solvent and water differ depending on a solubility ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) to beused. However, the solvents shown in the description of the reaction canbe used, in general. The ratio of organic solvent:water and molar ratioof the product (2) to water are also the same as those for theproduction reaction of the compound (2).

The formation of a pharmacologically acceptable salt can be easilycarried out by mixingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) with aninorganic acid such as hydrochloric acid, sulfuric acid or phosphoricacid, an organic acid such as acetic acid, maleic acid, fumaric acid,tartaric acid or citric acid, or an amino acid such as glycine orphenylalanine, in an aqueous liquid. The mixing method is notparticularly limited, and examples thereof are, for example, a method inwhich the above-mentioned inorganic acid, organic acid or amino acid isadded to an aqueous liquid containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2), a methodin which an aqueous liquid containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) is addedto an aqueous liquid of the above-mentioned inorganic acid, organic acidor amino acid, and the like.

The amount used of the above-mentioned inorganic acid, organic acid oramino acid is not particularly limited, and in general, when nosubstance having adverse influence exists, it may advantageously beapproximately a theoretical amount required based onN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2). In asolvent having a relatively high solubility for a pharmacologicallyacceptable salt of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-aminoacid (2), and the like, the equilibrium of salt formation can be shiftedby using an excess amount thereof to increase a deposition ratio of asalt. It is advantageous from economical viewpoint and the like to usethem, for example, in an amount of 0.9 to 2.5 fold, preferably 0.95 to2.0 fold, more preferably 0.95 to 1.2 fold of the theoretical amount.

When N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2)includes an isomer, for example, when the carbon atom in the formula (2)to which a ethoxycarbonyl group is connected is an asymmetric carbonatom, the above-mentioned inorganic acid, organic acid or amino acid canbe used in approximately the theoretical amount required, for example,in an amount of 0.9 to 1.2 fold, preferably 0.95 to 1.1 fold of thetheoretical amount based on the isomer having favorable antihypertensiveaction in which said carbon atom has (S)-configuration.

It is preferable to form the pharmacologically acceptable salt in anaqueous liquid essentially consisting of water because, in particular,effect for suppressing the production of a diketopiperazine derivative(3) is remarkably high, and contamination of an organic solvent which isnot preferable for a human body into the final product is avoided.However, it turns out that, when theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) isN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline and thepharmacologically acceptable salt thereof is maleic acid salt, there areproblems for the production in an industrial scale that high yield isnot easily obtained and property and condition of the produced crystaldeteriorate and adverse influence is exerted on filtration property anddrying property since the water-solubility of the maleate is rather higheven at low temperature. Furthermore, any satisfactory method to solvethe problems has not been known hitherto.

Then, it has been found that these problems can be favorably solved byadding and/or allowing to coexist an inorganic salt having highsalting-out effect, particularly sodium chloride, potassium chloride orthe like, by carrying out the steps of forming and crystallizing a saltwithin a range of 40 to 70° C., or by employing these methods alone orin combination thereof.

Especially when a pharmacologically acceptable salt is formed from areaction solution which is obtained by condensing an amino acid (1) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride inan aqueous liquid essentially consisting of water, the yield of thepharmacologically acceptable salt can be increased due to thesalting-out effect of an inorganic salt derived from a pH adjustingagent even without adding a further inorganic salt. Particularly, in thecase of a reaction solution obtained by carrying out condensation at ahigh reaction concentration of at least 10% (w/v), preferably at least20% (w/v), more preferably at least 30% (w/v), the concentration of theinorganic salt derived from a pH adjusting agent is necessarilyincreased and therefore higher salting-out effect can be expected.

When N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate(enalapril maleate) is formed in an aqueous liquid in which a largeamount of a water-soluble inorganic salt generated from the productionreaction of the compound (2) is dissolved, there is a tendency that thesolubility of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolineand maleic acid in the above-mentioned aqueous liquid decreases and thenthe formation of the maleate becomes incomplete, resulting in decreaseof the yield and remaining and contamination of maleic acid which is aninsoluble component. Further, the property and condition of theresulting crystal are generally poor, and adverse influence is exertedalso on filtration property and drying property. Thus, it is importantthat the concentration of an inorganic salt is not too high in the stepsof forming and crystallizing a maleate. The upper limit of the inorganicsalt concentration at the formation and crystallization of the saltcannot be stipulated unconditionally since it depends on theconcentration, temperature and method in operation and the kind of aninorganic salt and the like. The concentration is generally at most 15%by weight, preferably at most 10% by weight for suitable operation.

From the above-mentioned viewpoints, for preferably solving theseproblems, there can be used a method in which an aqueous liquid ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril) inwhich an inorganic salt coexists is gradually added to an aqueous liquidcontaining maleic acid. By this method, unexpectedly salt formation canbe carried out with excellent yield and quality without using a largeamount of maleic acid. According to this method, the crystallizationamount can be advantageously raised by enhancement of salting-out effectdue to phenomenon in which the inorganic salt concentration is low ininitial stage of a salt formation and the inorganic salt concentrationis finally increased. Though sufficient crystallization amount can beusually obtained by this operation, it is also possible to newly add aninorganic salt to increase the crystallization amount according todemands. Regarding removal of impurities,N-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-L-proline (7) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) can beadvantageously removed, and consequently,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline can beobtained having a low content of these impurities.

The time for addition of the aqueous liquid of enalapril in which aninorganic salt coexists is not particularly limited and, in general, thetime required for adding the whole amount thereof is at least ¼ hour,normally at least ⅓ hour, preferably at least ½ hour. The amount ofmaleic acid used is 0.9 to 3.0 molar equivalents, preferably 0.95 to 2.0molar equivalents, more preferably 0.95 to 1.2 molar equivalents, basedon enalapril.

The operation temperature for forming the pharmacologically acceptablesalt of the present invention depends on the kind of a solvent, the kindof a salt formed, an operation manner and the like, and cannot beparticularly limited, however, it is carried out preferably at 40 to 70°C., and it is suitably carried out more preferably at 50 to 70° C.,particularly about 60° C. A system in which water coexists isparticularly important since heating in a system in which no watercoexists leads to the production of the by-product diketopiperazinederivative (3). The salt formation at lower temperature is notpreferable because a fine crystal is deposited and slurry in the form ofwhip is formed to deteriorate fluidity and filterability, and theresulting crystal has a high liquid content and cannot be easily dried,and the like. Though these problems can be advantageously improved byraising the salt formation temperature as described above, it has beenfound that raising of the salt formation temperature is also preferablefor improving a property of removing impurities. As to an advantageousmethod for raising the salt formation temperature, the method in whichN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril) isgradually added to the aqueous liquid of maleic acid as described aboveis preferable also from a viewpoint that the thermal histeresis onenalapril before the salt formation can be reduced. Further, increase inthe proportion of water in an aqueous liquid used is also advantageoussince it further enhances the effect for suppressing the production ofthe by-product diketopiperazine derivative (3). Finally, the reactionmixture can be cooled to a temperature of at most 20° C., preferably atmost 10° C. to increase the amount crystallized.

Particularly, in a method for convertingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (enalapril) toits maleate, the amount crystallized ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate can beincreased by adding and/or allowing to coexist an inorganic salt such assodium chloride or potassium chloride in an aqueous liquid containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline and maleicacid. The aqueous liquid is preferably a medium comprising an organicsolvent and water in a weight ratio of from 96:4 to 0:100. As theabove-mentioned organic solvent, the one usable for the productionreaction of the compound (2) as described above can be suitably used.The amount of water to enalapril can be the same as that for theabove-mentioned compound (2). The amount of maleic acid is 0.9 to 3.0molar equivalents, preferably 0.95 to 2.0 molar equivalents, morepreferably 0.95 to 1.2 molar equivalents, based on enalapril.

Thus obtained pharmacologically acceptable salt ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) can be,if necessary, purified, for example, according to a method such asrecrystallization or reslurry washing. The reslurry washing means amethod in which a crystal is added to a medium and stirred in the formof slurry and filtrated for purification. A trace amount of a by-productdiketopiperazine derivative (3) is produced also from theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) which hasbeen stabilized as a salt, however, the production of diketopiperazinederivative (3) can be suppressed by carrying out this purificationoperation in an aqueous liquid. Namely, the existence of watersuppresses the production of a by-product diketopiperazine derivative(3) also in this purification operation.

Furthermore, as described above, for increasing the yield, the additionof an inorganic salt can be suitably carried out, and the operationtemperatures for recrystallization and reslurry washing can be raised,and preferably the operation can be carried out at a temperature in therange from 40 to 70° C., and can be suitably carried out more preferablyat from 50 to 70° C., particularly about 60° C., from viewpoint ofimproving the property and condition of the resulting slurry andcrystal, as described above.

By such operations, an N-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-aminoacid (4) and N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) can beadvantageously reduced and, consequently, anN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) having alow content of these impurities can be obtained. This impurity removingeffect differs depending on the purification method and the kind ofsolvent, and cannot be generally defined and, for example, the amount ofimpurities can be lowered to a level of at most {fraction (1/10)},preferably to a level at which the content in the resulting salt isnegligible, as described in Example 9 shown below. The above-mentionedaqueous liquid is preferably a medium comprising an organic solvent andwater in a weight ratio of 96:4 to 0:100. As the above-mentioned organicsolvent, that which can be used for the production reaction of thecompound (2) as described above can be suitably used.

The by-product diketopiperazine derivative (3) once produced by anoperation in a dehydrated solution does not easily regenerate theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) and apharmacologically acceptable salt thereof even if water is addedthereafter.

A most preferable embodiment of the present invention is a process forpreparing a pharmacologically acceptable salt ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) whichcomprises reacting an amino acid (1) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride inan aqueous liquid essentially consisting of water under theabove-mentioned conditions, subsequently forming a pharmacologicallyacceptable salt thereof from the reaction mixture containing theresulting N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid(2), without extracting the product (2).

Another most preferable embodiment of the present invention is a processfor preparing a pharmacologically acceptable salt ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2) whichcomprises reacting an amino acid (1) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride ina mixed solvent system of water and an organic solvent having a lowmiscibility with water or in an aqueous liquid essentially consisting ofwater under the above-mentioned specific conditions, extracting andseparating N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid(2) from the resulting reaction mixture to obtain a water-saturatedorganic solvent phase containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2), thenforming a pharmacologically acceptable salt thereof.

From thus obtained pharmacologically acceptable salt ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid (2), acrystal can be separated according to a method such as centrifugalseparation, pressure filtration or filtration under reduced pressure,and washed, and dried under normal pressure or reduced pressure.

The present invention is more specifically explained below by means ofExamples. It is to be understood that the present invention is notlimited only to those Examples.

The HPLC analysis was carried out under the following conditions:

Column: FINEPAK SIL C18-5(Trade name, 4.6 mm×25 cm, available from JASCOCORP.)

Eluent: 0.1 M KH₂PO₄(pH2.8)/CH₃CN (70:30(V/V))

Flow rate: 1.0 ml/min

Temperature: 45° C.

Detection condition: UV 210 nm

EXAMPLE 1

To 22.02 g (191 mmol) of L-proline were added 20 ml of ethyl acetate and22 ml of H₂O and, then, the pH was adjusted to 10.5 with 30% by weightNaOH aqueous solution. The inner temperature was regulated to 19 to 20°C. and, then, thereto was slowly added dropwise a solution containing29.20 g (96 mmol) of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride in 156 ml of ethyl acetate over 4 hours withstirring. During the dropping, the reaction mixture was maintained at apH of 10.5±0.5 with adding dropwise 30% by weight NaOH aqueous solution,and simultaneously, the inner temperature was maintained at 19 to 20° C.and the agitating power was maintained at 1 kW/m³. After completion ofthe dropping, the stirring was continued for 1 hour under the sameconditions. The inner temperature was raised to 30° C., and the pHthereof was adjusted to 4.5±0.2 with 35% by weight HCl. Under the sameconditions, the stirring was continued for 10 minutes to complete thedecarboxylation. The organic phase was separated at 30° C. and, then,the water phase was further extracted once with 20 ml of ethyl acetateat 30° C. The resulting organic phases were mixed and then washed oncewith H₂O in an amount of 5% by volume of the organic phase at 30° C. Thewater-saturated organic phase was analyzed with HPLC and, as a result, asolution containing 14% by weightN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was obtained.The reaction ratio was 97% and the extraction recovery was 96% (bothwere calculated by HPLC absolute calibration curve method. Hereinafterthe same). The amounts produced of the by-products were as follows:diketopiperazine derivative (3) 0.5% by weight, carboxy derivative (4)0.4% by weight, N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5)0.5% by weight.

Further, to this solution was added 10.49 g (90 mmol) of maleic acidwith stirring at an inner temperature of 30° C. The stirring wascontinued for 1 hour under the same conditions and, then, the innertemperature was cooled down to 5° C. over 3 hours, and the stirring wascontinued for further 2 hours. The deposited crystal was taken out byfiltration under reduced pressure, and washed twice with 80 ml of ethylacetate cooled to 5° C. The resulting wet crystal was dried underreduced pressure (20 to 50° C., 30→1 mmHg), to obtain 42.54 g (86 mmol)of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate.The purity was at least 99% and the contents of the diketopiperazinederivative (3), carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) were respectivelyat most 0.05% by weight. The yield fromN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxyanhydride was90%.

EXAMPLE 2

To 40 ml of H₂O was added 22.02 g (191 mmol) of L-proline and, then, thepH was adjusted to 10.5 with 30% by weight NaOH aqueous solution. Theinner temperature was regulated to 19 to 20° C. and, then, thereto wasslowly added 29.20 g (96 mmol) of a crystal ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydrideover 6 hours with stirring. During the addition, the reaction mixturewas maintained at a pH of 10.5±0.5 with adding dropwise 30% by weightNaOH aqueous solution, and simultaneously, the inner temperature wasmaintained at 19 to 20° C. and the agitating power was maintained at 1.2kW/m³. After completion of the addition, the stirring was continued for1 hour under the same conditions. The inner temperature was raised to30° C. and the pH thereof was adjusted to 4.2±0.2 with 35% by weightHCl. Then, under the same conditions, the stirring was continued for 10minutes to complete the decarboxylation. The reaction mixture wasanalyzed with HPLC and, as a result, a solution containing 21% by weightN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was obtained.The reaction ratio was 97% and the amounts produced of the by-productswere as follows: diketopiperazine derivative (3) at most 0.05% byweight, carboxy derivative (4) 0.4% by weight,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) 0.6% by weight.

This solution containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was added to asolution of 10.66 g (92 mmol) of maleic acid in 20 ml of H₂O, withstirring at an inner temperature of 60° C. over 1 hour. The stirring wascontinued for 1 hour under the same conditions and, then, the innertemperature was cooled down to 5° C. over 3 hours, and the stirring wascontinued for further 2 hours. The deposited crystal was taken out byfiltration under reduced pressure, and quickly washed three times with80 ml of H₂O cooled to 0 to 3° C. . The resulting crystal had a goodfilterability and a glossy and excellent crystal form; In the case offorming the salt at an inner temperature of 30° C., the resulting saltwas slurry in the form of whip and had a poor filterability and a formof fine crystal. The resulting wet crystal was dried under reducedpressure (20 to 50° C., 30→1 mmHg), to obtain 42.05 g (85 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate. Thepurity was at least 99% and the contents of the diketopiperazinederivative (3), carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) were respectivelyat most 0.05% by weight. The yield fromN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine N-carboxyanhydride was89%.

EXAMPLE 3

To 22 ml of H₂O was added 22.02 g (191 mmol) of L-proline and, then, tothis L-proline aqueous solution was added a solution containing 29.20 g(96 mmol) of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride in 176 ml of ethyl acetate. The inner temperature wasregulated to 19 to 20° C. and, then, the reaction mixture was maintainedat a pH of 10.5±1.0 with adding dropwise 30% by weight NaOH aqueoussolution for 5 hours under stirring, and simultaneously, the agitatingpower was maintained at 1 kW/m3. The inner temperature was raised to 30°C. and the pH thereof was adjusted to 4.5±0.2 with 35% by weight HCl.Then, under the same conditions, the stirring was continued for 10minutes to complete the decarboxylation. The organic phase was separatedat 30° C. and, then, the water phase was further extracted once with 20ml of ethyl acetate at 30° C. . The organic phases were mixed and, thenwashed once with H₂O in an amount of 5% by volume of the organic phaseat 30° C. The water-saturated organic phase was analyzed with HPLC and,as a result, a solution containing 14% by weightN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was obtained.The reaction ratio was 96% and the extraction ratio was 96%. The amountsproduced of the by-products were as follows: diketopiperazine derivative(3) 0.4% by weight, carboxy derivative (4) 0.5% by weight,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) 0.6% by weight.

Further, to this solution was added a solution of 10.25 g (88 mmol) ofmaleic acid in 20 ml of H₂O, with stirring at an inner temperature of30° C. The stirring was continued for 1 hour under the same conditionsand, then, the inner temperature was cooled down to 5° C. over 3 hours,and the stirring was continued for further 2 hours. The depositedcrystal was taken out by filtration under reduced pressure, and washedtwice with 80 ml of ethyl acetate cooled to 5° C. The resulting wetcrystal was dried under reduced pressure (20 to 50° C., 30→1 mmHg), toobtain 41.67 g (85 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate. Thepurity was at least 99% and the contents of the diketopiperazinederivative (3), carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) were respectivelyat most 0.05% by weight. The yield fromN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride was88%.

EXAMPLE 4

To 22.02 g (191 mmol) of L-proline and 29.20 g 96 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride wasadded 60 ml of H₂O. The inner temperature was regulated to 19 to 20° C.and, then, the reaction mixture was maintained at a pH of 10.5±1.0 withadding dropwise 30% by weight NaOH aqueous solution for 6 hours understirring, and simultaneously, the agitating power was maintained at 0.9kW/m³. The inner temperature was raised to 30° C. and the pH thereof wasadjusted to 4.2±0.2 with 35% by weight HCl. After adjusting of pH, underthe same conditions, the stirring was continued for 10 minutes tocomplete the decarboxylation. The reaction mixture was analyzed withHPLC and, as a result, a solution containing 18% by weightN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was obtained.The reaction ratio was 96%, and the amounts produced of the by-productswere as follows: diketopiperazine derivative (3) at most 0.05% byweight, carboxy derivative (4) 0.4% by weight,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) 0.7% by weight.

To this solution was added a solution wherein 21.09 g (182 mmol) ofmaleic acid was dissolved in 40 ml of H₂O, with stirring at an innertemperature of 30° C. The stirring was continued for 1 hour under thesame conditions and, then, the inner temperature was cooled down to 5°C. over 3 hours, and the stirring was continued for further 2 hours. Thedeposited crystal was taken out by filtration, and quickly washed twicewith 20 ml of H₂O cooled to 0 to 3° C. The resulting wet crystal wasdried under reduced pressure (20 to 50° C., 30→1 mmHg), to obtain 41.29g (84 mmol) of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinemaleate. The purity was at least 99% and the contents of thediketopiperazine derivative (3), carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) were respectivelyat most 0.05% by weight. The yield fromN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride was87%.

EXAMPLE 5

To 60 ml of H₂O was added 22.02 g (191 mmol) of L-proline and, then,25.47 g (191 mmol) of 30% by weight NaOH aqueous solution was addedthereto with stirring. At this time, the pH of the solution is 12.9. Theinner temperature was regulated to 14 to 15° C. and, then, thereto wasadded 29.20 g (96 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydrideover 30 minutes with stirring. After the completion of the addition, thepH of the reaction mixture became approximately 10 in 30 minutes, andfor 8 hours from this time, the reaction mixture was maintained at a pHof 10.0±0.5 with adding dropwise 30% by weight NaOH aqueous solution,and simultaneously, the agitating power was maintained at 0.9 kW/m³ tocomplete the reaction. With the inner temperature maintained at 15° C.,the pH was adjusted to 4.0±0.2 with 35% by weight HCl. After adjustingthe pH, under the same conditions, the stirring was continued for 10minutes to complete the decarboxylation. The reaction mixture wasanalyzed with HPLC and, as a result, a solution containing 18% by weightN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was obtained.The reaction ratio was 99% and the amounts produced of the by-productswere as follows: diketopiperazine derivative (3) 0.05% by weight,carboxy derivative (4) 0.1% by weight,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) 0.6% by weight.

This solution containingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was added to asolution. prepared by dissloving 11.31 g (97 mmol) of maleic acid in 20ml of H₂O, with stirring at an inner temperature of 60° C. over 1 hour.The stirring was continued for 1 hour under the same conditions and,then, the inner temperature was cooled down to 5° C. over 3 hours, andthe stirring was continued for further 1 hour. The deposited crystal wastaken out by filtration, and quickly washed twice with 80 ml of H₂Ocooled to 0 to 3° C. The resulting crystal had a good filterability, aglossy and excellent crystal form; In the case of forming the salt at aninner temperature of 30° C., the resulting salt was slurry in the formof whip and had a poor filterability and a form of fine crystal. Theresulting wet crystal was dried under reduced pressure (20 to 50° C.,30→1 mmHg), to obtain 42.52 g (86 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate. Thepurity was at least 99% and the contents of the diketopiperazinederivative (3), carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) were respectivelyat most 0.05% by weight. The yield fromN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride was90%.

EXAMPLE 6

According to Example 1, using various solvents shown in Table 1,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was preparedand applicability of the various solvents was examined. The reactionratios in various solvents are shown in Table 1.

TABLE 1 Miscibility of Reaction organic solvent ratio with waterSolvents (%) Low Ethyl acetate/H₂O 98 Methylene chloride/H₂O 96 Methylisobutyl ketone/H₂O 95 Methyl isopropyl ketone/H₂O 95 High CH₃CN/H₂O 95THF/H₂O 96 — H₂O 97

EXAMPLE 7

According to Example 1 except that the pH was changed as shown in Table2, N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline wassynthesized and the influence of pH was investigated. The reaction ratioat each pH and the amounts produced (% by weight) of thediketopiperazine derivative (3),N-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-L-proline (carboxy derivative(4)) and N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (ALE (5)) areshown in Table 2. The control of pH was within the range of ±1.0.

TABLE 2 Amount Amount produced of Amount produced of produced Reactiondiketopiperazine carboxy derivative of ALE ratio derivative (3) (4) (5)(% pH (%) (% by weight) (% by weight) by weight) 8.0 84 0.3 0.0 9.5 9.095 0.4 0.0 4.2 10.5 98 0.5 0.4 0.6 12.0 95 0.5 1.5 0.3 13.0 90 0.4 5.50.2

EXAMPLE 8

According to Example 1, an ethyl acetate solution ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was prepared.129.40 Grams of this organic phase was washed twice with 30 ml ofsaturated brine, and dehydrated with magnesium sulfate. The solvent wasconcentrated and, then, the concentrated liquid was dried under reducedpressure to obtain an oily reaction product. This product was dissolvedin ethyl acetate having a water content shown in Table 3 or water toprepare a solution having a concentration of about 13% by weight, andthe solution was warmed to 30° C. and stirred for 6 hours. The amountproduced of the diketopiperazine derivative (3) was analyzed with HPLC,and the relation between water content and the average increase in thediketopiperazine derivative (3) per hour was investigated. The resultsare shown in Table 3. The molar ratio in Table 3 means mol number ofwater per 1 mol ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline.

TABLE 3 Average increase Water content per hour (% by weight) (molarratio) (% by weight/hr) 0.1 0.1 2.85 0.4 0.6 2.06 1.1 1.6 1.32 2.1 3.10.99 3.8 6.1 0.48 5.1 8.2 0.39 100 160 0.02

EXAMPLE 9

According to Example 1,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate wasprepared. Thereto was added ethyl acetate containing 0.1% water or H₂Oto prepare a solution having a concentration of 16% by weight, and thesolution was warmed to 60° C. and stirred for 6 hours. The amountproduced of the diketopiperazine derivative (3) was analyzed with HPLC,and the relation between water content and the average increase in thediketopiperazine derivative (3) per hour was investigated. The resultsare shown in Table 4.

TABLE 4 Average increase per hour Solvents (% by weight/hr) Ethylacetate 0.55 containing 0.1% water H₂O 0.06

EXAMPLE 10

To 5.00 g (28.2 mmol) of 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid were added 20 ml of ethyl acetate and 10 ml of H₂O and, then, thepH was adjusted to 10.5 with 30% by weight NaOH aqueous solution. Theinner temperature was regulated to 20° C. and, then, 4.31 g (14.1 mmol)of a crystal of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride was slowly added over 5 hours with stirring. Duringthe addition, the reaction mixture was maintained at a pH of 10.5±1.0with adding dropwise 30% by weight NaOH aqueous solution, andsimultaneously, the inner temperature was maintained at 20° C. and theagitating power was maintained at 1.3 kW/m³. After completion of theaddition, the stirring was continued for 2 hour under the sameconditions. The mixture was cooled to 5° C., and the pH thereof wasadjusted to 4.5±0.2 with 35% by weight HCl. Then, under the sameconditions, the stirring was continued for 10 minutes to complete thedecarboxylation. The deposited material was removed by filtration underreduced pressure, and the organic phase was separated at 5° C. and thewater phase was further extracted once with 20 ml of ethyl acetate at10° C. . The resulting organic phases were mixed and then washed threetimes with H₂O in an amount of 5% by volume of the organic phase at 5°C. The organic phase was concentrated under reduced pressure to obtain6.43 g of an oil. The oil was dissolved in ethyl acetate having a watercontent shown in Table 5 to prepare a solution having a concentration ofabout 12% by weight, and the resulting solution was stirred for 6 hoursat 10° C. The amount produced of the diketopiperazine derivative (3) wasanalyzed with HPLC, and the relation between water content and theaverage increase in the diketopiperazine derivative (3) per hour wasinvestigated. The results are shown in Table 5.

TABLE 5 Average increase Water content per hour (% by weight) (% byweight/hr) 0.1 5.3 1.0 2.2 2.1 1.7 3.8 1.3

EXAMPLE 11

According to Example 1, an ethyl acetate solution ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was prepared.To 50 ml of the ethyl acetate solution was added H₂O in an amount of 10%by volume (based on ethyl acetate). The mixture was stirred for 10minutes at each temperature shown in Table 6, and allowed to stand stillfor 10 minutes and, then, separated. The amount ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline in thewater-saturated organic phase was analyzed with HPLC, and the relationbetween the temperature and the partition ratio to the organic phase wasinvestigated. The results are shown in Table 6.

TABLE 6 Temperature Partition ratio (° C.) to organic phase (%) 5 26 1035 20 57 25 72 30 88 40 94

EXAMPLE 12

To 22.02 g (191 mmol) of L-proline were added 20 ml of ethyl acetate and22 ml of H₂O and, then, the pH was adjusted to 10.5 with 30% by weightNaOH aqueous solution. The inner temperature was regulated to 19 to 20°C. and, then, thereto was slowly added dropwise a solution containing29-20 g (96 mmol) of N-phenylpropyl )-L-alanine. N-carboxyanhydrine in156 ml of ethyl acetate over 4 hours with stirring. During the dropping,the reaction mixture was maintained at a pH of 10.5±1.0 with addingdropwise 30% by weight NaOH aqueous solution, and simultaneously, theinner temperature was maintained at 19 to 20° C. and the agitating powerwas maintained at 0.7 kW/m³. After completion of the dropping, thestirring was continued for 1 hour under the same conditions. The innertemperature was raised to 30° C., and the pH thereof was adjusted to4.5±0.2 with 35% by weight HCl. Under the same conditions, the stirringwas continued for 10 minutes to complete the decarboxylation. Theorganic phase was separated at 30° C. and, then, the water phase wasextracted once with 20 ml of ethyl acetate at 30° C. The resultingorganic phases were mixed and cooled down to 0 to 3° C. and, then,back-extracted with 250 ml of H₂O. The reaction ratio was 98% and theextraction recovery was 94%. The amounts produced of the by-productswere as follows: diketopiperazine derivative (3) 0.3% by weight, carboxyderivative (4) 0.6% by weight,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) 0.4% by weight.

The obtained 12% by weight aqueous solution ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was warmed toan inner temperature of 30° C. and thereto was added 10.73 g (93 mmol)of maleic acid with stirring. The stirring was continued for 1 hourunder the same conditions and, then, the inner temperature was cooleddown to 5° C. over 3 hours. To the mixture was added 62.05 g of NaCl andthe stirring was continued for further 2 hours. The deposited crystalwas taken out by filtration under reduced pressure, and quickly washedthree times with 80 ml of H₂O cooled to 0 to 3° C. The resulting wetcrystal was dried under reduced pressure (20 to 50° C., 30→1 mmHg) toobtain 40.35 g (82 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate. Thepurity was at least 99% and the contents of the diketopiperazinederivative (3), carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) were respectivelyat most 0.05% by weight. The yield fromN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride was86%.

EXAMPLE 13

To 22.02 g (191 mmol) of L-proline were added 20 ml of ethyl acetate and22 ml of H₂O and, then, the pH was adjusted to 10.5 with 30% by weightNaOH aqueous solution. The inner temperature was regulated to 19 to 20°C. and, then, thereto was slowly added dropwise a solution containing29.20 g (96 mmol) of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride in 156 ml of ethyl acetate over 4 hours withstirring. During the dropping, the reaction mixture was maintained at apH of 10.5±0.5 with adding dropwise 30% by weight NaOH aqueous solution,and simultaneously, the inner temperature was maintained at 19 to 20° C.and the agitating power was maintained at 0.5 kW/m³. After completion ofthe dropping, the stirring was continued for 1 hour under the sameconditions. The inner temperature was raised to 30° C., and the pHthereof was adjusted to 4.5±0.2 with 35% by weight HCl. Under the sameconditions, the stirring was continued for 10 minutes to complete thedecarboxylation. The reaction ratio were 98% and the amounts produced ofthe by-products were as follows: diketopiperazine derivative (3) 0.5% byweight, carboxy derivative (4) 0.4% by weight,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) 0.6% by weight.

This two-phase liquid was stirred at an inner temperature of 30° C. andthereto was added 10.88 g (94 mmol) of maleic acid. The stirring wascontinued for 1 hour under the same conditions and, then, the innertemperature was cooled down to 5° C. over 3 hours, and the stirring wascontinued for further 2 hours. The deposited crystal was taken out byfiltration under reduced pressure, and washed once with 80 ml of H₂Ocooled to 0 to 30° C. and once with 80 ml of ethyl acetate cooled to 5°C. The resulting wet crystal was dried under reduced pressure (20 to 50°C., 30→1 mmHg) to obtain 41.54 g (84 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate. Thepurity was at least 99% and the contents of the diketopiperazinederivative (3), carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) were respectivelyat most 0.05% by weight. The yield fromN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride was86%.

EXAMPLE 14

To 22.02 g (191 mmol) of L-proline were added 20 ml of methylenechloride and 22 ml of H₂O and, then, the pH was adjusted to 10.5 with30% by weight NaOH aqueous solution. The inner temperature was regulatedto 19 to 20° C. and, then, thereto was slowly added dropwise a solutionprepared by dissolving 29.20 g (96 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride in156 ml of methylene chloride, over 4 hours with stirring. During thedropping, the reaction mixture was maintained at a pH of 10.5±1.0 withadding dropwise 30% by weight NaOH aqueous solution, and simultaneously,the inner temperature was maintained at 19 to 20° C. and the agitatingpower was maintained at 1 kW/m³. After completion of the dropping, thestirring was continued for 1 hour under the same conditions. The innertemperature was raised to 30° C., and the pH thereof was adjusted to4.5±0.2 with 35% by weight HCl. Under the same conditions, the stirringwas continued for 10 minutes to complete the decarboxylation. Theorganic phase was separated at 25° C. and, then, the water phase wasextracted once with 20 ml of methylene chloride at 25° C. The resultingorganic phases were mixed and then washed once with H₂O in an amount of5% by volume of the organic phase at 25° C. The water-saturated organicphase was analyzed with HPLC and, as a result, a solution containing 10%by weight N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline wasobtained. The reaction ratio was 97% and the extraction recovery was 99%The amounts produced of the by-products were as follows:diketopiperazine derivative (3) 0.6% by weight, carboxy derivative (4)0.5% by weight, N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5)0.5% by weight.

Further, to this solution was added 10.87 g (94 mmol) of maleic acidwith stirring at an inner temperature of 30° C. The stirring wascontinued for 1 hour under the same conditions and, then, the innertemperature was cooled down to 5° C. over 3 hours, and the stirring wascontinued for further 2 hours. The deposited crystal was taken out byfiltration under reduced pressure, and washed once with 80 ml ofmethylene chloride cooled to 5° C. The resulting wet crystal was driedunder reduced pressure (20 to 50° C., 30→1 mmHg) to obtain 43.41 g (88mmol) of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinemaleate. The purity was at least 99% and the contents of thediketopiperazine derivative (3), carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) were respectivelyat most 0.05% by weight. The yield fromN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride was92%.

EXAMPLE 15

According to Example 2, using 30% KOH aqueous solution instead of 30%NaOH aqueous solution,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was obtained.The reaction ratio was 96% and the amounts produced of the by-productswere as follows: diketopiperazine derivative (3) at most 0.05% byweight, the carboxy derivative (4) 0.4% by weight andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) 0.6% by weight.

Further, according to Example 2, using the increased amount of maleicacid to 13.85 g (119 mmol) from 10.66 g (92 mmol),N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate wasobtained. The purity was at least 99% and the contents of thediketopiperazine derivative (3), carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) were respectivelyat most 0.05% by weight. The yield fromN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride was90%.

EXAMPLE 16

According to Example 12 except for omitting the addition of NaCl,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate wasobtained. The salting-out effect of NaCl was examined as compared within Example 12. The results are shown in Table 7. The crystallizationyield in Table 7 means a proportion of the crystallized maleate based onN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline, calculated bymole.

TABLE 7 Crystallization yield Solvents (%) Aqueous solution 83 Aqueoussolution + NaCl 93 (Example 12)

EXAMPLE 17

To 20.0 g (40.6 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleatecontaining 3.0% by weightN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) was added 300 ml ofH₂O and, then, this mixture was heated to 60° C. to be dissolved. Themixture was cooled to 5° C. over 4 hours with stirring, and thereto wasadded 38 g of NaCl and the stirring was continued for further 1 hour.The deposited material was taken out by filtration and washed twice with100 ml of H₂O cooled to 0 to 3° C. The resulting crystal had a goodfilterability and a glossy and excellent crystal form. The resulting wetcrystal was dried under reduced pressure (20 to 50° C., 30→1 mmHg) toobtain 17.2 g (34.9 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate. Thepurity was at least 99% and the content of theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) was at most 0.05%by weight. During the operation, the production of the by-productdiketopiperazine derivative (3) was not recognized.

EXAMPLE 18

To 20.0 g (40.6 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleatecontaining 3.0% by weightN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) was added 300 ml ofH₂O and, then, this mixture was warmed to 30° C. The mixture was cooledto 5° C. over 4 hours with stirring, and thereto was added 38 g of NaCland the stirring was continued for further 1 hour. The depositedmaterial was taken out by filtration and washed twice with 100 ml of H₂Ocooled to 0 to 3° C. The resulting wet crystal was dried under reducedpressure (20 to 50° C., 30→1 mmHg) to obtain 17.2 g (34.9 mmol) ofN-(i(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate. Thepurity was at least 99% and the content of theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) was at most 0.05%by weight. During the operation, the production of the by-productdiketopiperazine derivative (3) was not recognized.

EXAMPLE 19

To 20.0 g (40.6 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleatecontaining 3.0% by weightN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) was added 300 ml ofH₂O and, then, this mixture was kept at 5° C. for 4 hours with stirring.Thereto was added 38 g of NaCl and the stirring was continued forfurther 1 hour. The deposited material was taken out by filtration andwashed twice with 100 ml of H₂O cooled to 0 to 3° C. The resulting wetcrystal was dried under reduced pressure (20 to 50° C., 30→1 mmHg) toobtain 17.2 g (34.9 mmol) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline maleate. Thepurity was at least 99% and the content of theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) was at most 0.05%by weight. During the operation, the production of the by-productdiketopiperazine derivative (3) was not recognized.

COMPARATIVE EXAMPLE 1

To 10.0 g (27.9 mmol) of a diketopiperazine derivative (6) ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline were added 50ml of ethyl acetate and 50 ml of H₂O, and the mixture was stirred at 60°C. for 8 hours. The production ofN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline was notrecognized.

INDUSTRIAL APPLICABILITY

According to the present invention, anN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid and apharmacologically acceptable salt thereof having high quality can besimply and advantageously prepared with high yield and economicalefficiency.

Concretely, N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-amino acid(2) can be obtained with high yield because the production of theby-product diketopiperazine derivative (3) is suppressed by carryingout, in an aqueous liquid, a series of operations of from production ofthe compound (2) to formation of a pharmacologically acceptable saltthereof and, if necessary, isolation of the salt. The present inventiondoes not require to replace a solvent because the above-mentioned seriesof operations can be carried out in the same solvent and, therefore, theoperation can simply be carried out.

Further, there can be obtained the compound (2) having a low content ofa carboxy derivative (4) andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine (5) as well as adiketopiperazine derivative (3), by carrying out the reaction forproducing the compound (2) under a specific condition.

Additionally, N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinecan be efficiently separated by carrying out extraction and separationoperations under the specific temperature condition.

What is claimed is:
 1. A process for preparingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline whichcomprises producingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline from L-prolineand N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride, wherein condensation is carried out by graduallyadding at least one of N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride and a basic pH adjusting agent to an aqueous liquidcontaining L-proline and, if necessary,N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride,with the pH of the aqueous liquid maintained within a range of from 9 to12, and then decarboxylation is carried out under between neutral andacidic condition to obtainN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline having a lowcontent of a diketopiperazine derivative represented by a formula (6):

wherein all asymmetric carbon atoms with * have (S)-configuration,N-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-L-proline represented by aformula (7):

wherein all asymmertric carbon atoms with * have (S)-configuration, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine represented by aformula (5):


2. The process of claim 1 wherein at least one of theN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride andthe basic pH adjusting agent is added over at least ¼ hour.
 3. Theprocess of claim 1 or 2, wherein at least 2 molar equivalents ofL-proline is used based onN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride. 4.A process for preparingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline whichcomprises producingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline from L-prolineand N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride, wherein a reaction is started by addingN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride toan aqueous liquid containing at least 2 molar equivalents of L-prolineconstituting a basic salt based onN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine. N-carboxyanhydride,and after the pH of the aqueous liquid reaches a range of from 9 to 12 abasic pH adjusting agent is gradually added to the aqueous liquid tocarry out a condensation with the pH maintained within a range of from 9to 12, and then decarboxylation is carried out under between neutral andacidic condition to obtainN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline having a lowcontent of a diketopiperazine derivative represented by a formula (6):

wherein all asymmetric carbon atoms with * have (S)-configuration,N-(1(S)-carboxy-3-phenylpropyl)-L-alanyl-L-proline represented by aformula (7):

wherein all asymmetric carbon atoms with * have (S)-configuration, andN-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline represented bya formula (5):


5. The process of claim 1, 2 or 4 wherein the aqueous liquid comprisesan organic solvent and water in a weight ratio of from 96:4 to 0:100. 6.The process of claim 1, 2, or 4 wherein, in the reaction of L-prolineand N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanine.N-carboxyanhydride, stirring and mixing are carried out at an agitatingpower of at least 0.1 kW/m³.
 7. The process of claim 5 wherein theorganic solvent is at least one member selected from the groupconsisting of a halogenated hydrocarbon, a fatty acid ester, a ketoneand an ether.
 8. The process of claim 1, 2 or 4 wherein the reaction ofproducing N-(1(S)-ethoxy-carbonyl-3-phenylpropyl)-L-alanyl-L-proline iscarried out in an aqueous liquid essentially consisting of water.